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Production of donor-derived sperm after spermatogonial stem cell transplantation in the dog

James A. Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA¹ Center for Animal Resources and Education, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA² Center for Animal Transgenesis and Germ Cell Research, School of Veterinary Medicine, University of Pennsylvania, Kennett Square, Pennsylvania 19348, USA³ Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853, USA

Correspondence should be addressed to A J Travis; Email: ajt32{at}cornell.edu

Spermatogonial stem cell transplantation (SSCT) offers unique approaches to investigate SSC and to manipulate the male germline. We report here the first successful performance of this technique in the dog, which is an important model of human diseases. First, we investigated an irradiation protocol to deplete endogenous male germ cells in recipient testes. Histologic examination confirmed >95% depletion of endogenous spermatogenesis, but retention of normal testis architecture. Then, 5-month-old recipient dogs (n=5) were focally irradiated on their testes prior to transplantation with mixed seminiferous tubule cells (fresh (n=2) or after 2 weeks of culture (n=3)). The dogs receiving cultured cells showed an immediate allergic response, which subsided quickly with palliative treatment. No such response was seen in the dogs receiving fresh cells, for which a different injection medium was used. Twelve months post-injection recipients were castrated and sperm was collected from epididymides. We performed microsatellite analysis comparing DNA from the epididymal sperm with genomic DNA from both the recipients and the donors. We used six markers to demonstrate the presence of donor alleles in the sperm from one recipient of fresh mixed tubule cells. No evidence of donor alleles was detected in sperm from the other recipients. Using quantitative PCR based on single nucleotide polymorphisms (SNPs), about 19.5% of sperm were shown to be donor derived in the recipient. Our results demonstrate the first successful completion of SSCT in the dog, an important step toward transgenesis through the male germline in this valuable biomedical model.

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